Eukaryotic cell culture was first achieved in the early 1950s. Since that time, a wide range of transformed and primary cells have been cultivated using a wide variety of media and defined supplements such as growth factors and hormones as well as undefined supplements such as sera and other body extracts. For example, fibroblasts taken from a part of an animal such as the skin, can be routinely cultivated through many cell generations as karyotypically diploid cells or indefinitely as established cell lines. Epithelial cells however have morphological and proliferative properties that differ from fibroblasts are more difficult to cultivate. Indeed, in vitro, epithelial cells are commonly overgrown by fibroblasts when the two cells are grown together.
A diverse range of media have been developed for growing epithelial cells in a clonally competent manner. In some cases these cells can produce at least partially differentiated epithelium. Approaches to cultivation of epithelial cells in particular skin epithelia (keratinocytes) have included the following: cultivating cells on a feeder layer of lethally irradiated fibroblasts (Rheinhardt et al. 1975, Cell 6:331-343); cultivating keratinocytes on semi-synthetic collagen matrices (Eisenberg 1994, U.S. Pat. No. 5, 282,859; Bell 1990, EP 0361957); adding biological extracts including pituitary extracts and sera to specialized media; and utilizing a range of growth supplements including epidermal growth factor, and insulin (Boisseau et al. 1992, J. Dermatol. Sci. 3(2):111-120; Willie 1994, U.S. Pat. No. 5,292,655).
The skin.
Numerous attempts have been described for growing epithelial cells in such a way as to mimic human skin for purposes of wound treatment, in particular treatment of burns. The skin consists of two types of tissue. These are: (1) the stroma or dermis which includes fibroblasts that are loosely dispersed within a high density collagen matrix as well as nerves, blood vessels and fat cells; (2)the epidermis which includes an epidermal basal layer of tightly packed, actively proliferating immature epithelial cells. As the cells of the basal layer replicate, some of the young cells remain in the basal layer while others migrate outward, increase in size and eventually develop an envelop resistant to detergents and reducing agents. In humans, a cell born in the basal layer takes about 2 weeks to reach the edge or outer layer after which time the cells die and are shed. The skin contains various structures including hair follicles, sebaceous glands and sweat glands. Hair follicles are formed from differentiating keratinocytes that densely line invaginations of the epidermis. The open ended vesicles that formed from such invaginations collect and concentrate the secreted keratin and a hair filament results. Alternatively, epidermal cells lining an invagination may secrete fluids (sweat gland) or sebum (sebaceous gland). The regulation of formation and proliferation of these structures is unknown.
The constant renewal of healthy skin is accomplished by a balanced process in which new cells are being produced and aged cells die. There is a need to understand how this precise regulation comes about in order to counteract abnormal events occurring in aging, and also through disease and trauma that disrupt the balance. For example, psoriatic cells proliferate and die at an accelerated pace taking only about 15% of the time normally observed. Epidermal neoplasia arises when the epidermal cells multiply without control and rapidly overtake the number of cells normally dying. In chronic wounds, normal epidermal and dermal regeneration fails to occur.
Cultivation of skin in vitro.
Numerous attempts at growing skin in vitro have been undertaken. These attempts almost all include the step of separating the keratinocytes in the epidermis from fibroblasts and fat cells in the dermis. Where separation of keratinocytes is not performed, whole organs have been used. Attempts to cultivate organs in vitro have been limited to incubating organs in a serum containing medium (Li et al. 1991, Proc. Natl. Acad. Sci. 88(5):108-112). Where isolation of keratinocytes is performed, these cells are grown in a manner that permits the formation of a stratified epidermis. The epidermis prepared in this manner lacks hair follicles and sweat glands and the natural relationship between the epidermis and the dermis is not preserved. Cultivation methods including growing keratinocytes on non viable fibroblasts (Rheinwald et al. 1975, Cell 6:331-343); or placing the keratinocytes from the animal on a dermal substrate of collagen and fibroblasts that is synthetic or has been derived from an alternative source from that of the epidermis (Sugihara et al. 1991, in vitro Cell Dev. Biol. 27:142-146; Parenteau et al. 1991, J. Cell Biochem. 45(3):245-251).
Most existing in vitro models of the epidermis lack hair follicles, sweat glands and sebaceous glands (for a review of epidermal cell culture, see Coulomb et al. 1992, Pathol. Biol. Paris, 40(2):139-146). Exceptions include the gel-supported skin model of Li et al. (1991) who utilized skin explants with dimensions of 2.times.5 mm.sup.2 and 2.0 mm thick that remained viable for several days in the presence of serum containing media.
It would be desirable to have an in vitro model of the skin in a serum free environment where the natural intercellular relationships that occur in vivo are maintained so as to more accurately study how skin is formed and remains viable. For example, insights into how hair follicle formation occurs would have significant therapeutic applications including treatment for balding men, for patients undergoing chemotherapy and for skin grafting.
An in vitro model of the skin that closely mimics the properties of skin in vivo would have utility in screening assays in which compounds could be tested for their ability to repair or damage the skin (Kao et al. 1985, Toxicol. Appl. Pharmacol. 81:502-516; Goldberg ed. 1989, Alternative Methods in Toxicology, vol. 7, pp. v-vi, New York:Liebert). Requirements of a reproducible model for screening might include consistency in tissue architecture and nutritional environment in vitro, as well as prolonged viability and proliferation of cultures beyond 24 hours to observe threshold effects of compounds being screened. This level of consistency cannot be achieved in the presence of undefined media supplements such as sera or tissue extracts that vary between batches and cannot be adequately controlled. The dependence of a model on external growth supplements such as growth factors is also undesirable as growth factors or hormones may be included among the compounds to be tested. At present, existing in vitro skin models either require exogenous factors (Boyce et al. 1983, J. Invest. Dermatol. 81:33-4) or serum (Li et al. 1991, Proc. Natl. Acad. Sci. 88(5):108-112) or are only viable for short periods of time.
Epithelia.
The skin is one example of an epithelial tissue supported by stromal tissue containing fibroblasts. Epithelial tissues are found in every part of the body where an interface between an organ and the environment arise. Epithelial cells cycle continuously in an uninjured body and form the covering tissue for all the free surfaces in the body including the skin. In some cases, such as in the pancreas, the epithelial cells line numerous invaginations and secrete enzymes into open spaces that enable the organ to function. The lung is another example of a highly invaginated organ, each invagination in the lung being lined with epithelial cells through which air diffuses from the environment in to the body. Once again, these epithelial ells have characteristic properties. The lining of the gut is also composed of specialized epithelial cells that not only form a barrier but contain specialized structures for selectively absorbing food. All the epithelia are supported by a stroma of connective tissue.
There is a need for in vitro methods of culturing and maintaining organ cultures in which the cells preserve their naturally occurring intracellular relationships for extended periods of time. The availability of a tissue model in which differentiation, cell proliferation and cell homeostasis occurs would have utility in understanding the mechanisms by which organs are maintained in a healthy state and consequently how abnormal events may be reversed.